Inhibition of Gene Markers of Fibrosis with a Novel Inhibitor of Transforming Growth Factor-β Type I Receptor Kinase in Puromycin-Induced Nephritis

Abstract

SB-525334 (6-[2-tert-butyl-5-(6-methyl-pyridin-2-yl)-1H-imidazol-4-yl]-quinoxaline) has been characterized as a potent and selective inhibitor of the transforming growth factor-␤1 (TGF-␤1) receptor, activin receptor-like kinase (ALK5). The compound inhibited ALK5 kinase activity with an IC 50 of 14.3 nM and was ϳ4-fold less potent as an inhibitor of ALK4 (IC 50 ϭ 58.5 nM). SB-525334 was inactive as an inhibitor of ALK2, ALK3, and ALK6 (IC 50 Ͼ 10,000 nM). In cell-based assays, SB-525334 (1 M) blocked TGF-␤1-induced phosphorylation and nuclear translocation of Smad2/3 in renal proximal tubule cells and inhibited TGF-␤1-induced increases in plasminogen activator inhibitor-1 (PAI-1) and procollagen ␣1(I) mRNA expression in A498 renal epithelial carcinoma cells. In view of this profile, SB-525334 was used to investigate the role of TGF-␤1 in the acute puromycin aminonucleoside (PAN) rat model of renal disease, a model of nephritis-induced renal fibrosis. Orally administered doses of 1, 3, or 10 mg/kg/day SB-525334 for 11 days produced statistically significant reductions in renal PAI-1 mRNA. Also, the compound produced dose-dependent decreases in renal procollagen ␣1(I) and procollagen ␣1(III) mRNA, which reached statistical significance at the 10-mg/kg/day dose when compared with vehicle-treated PAN controls. Furthermore, PAN-induced proteinuria was significantly inhibited at the 10-mg/kg/day dose level. These results provide further evidence for the involvement of TGF-␤1 in the profibrotic changes that occur in the PAN model and for the first time, demonstrate the ability of a small molecule inhibitor of ALK5 to block several of the markers that are predictive of fibrosis and renal injury in this model.

Inhibition of ALK5 Signaling Induces Physeal Dysplasia in Rats

“…This lack of cross-reactivity serves to demonstrate that the physeal effects were a function of ALK inhibition at toxicologic, suprapharmacologic doses rather than off-target pharmacology. Closely related ALK5 inhibitors were previously assayed for effects on other ALK family members (Inman, 2002;Grygielko, 2005). These inhibitors were approximately 10-20-fold less potent as inhibitors of ALK4 and ALK7, and 100-fold less potent as inhibitors of ALK3 and ALK6.…”

“…Compounds were also evaluated in the purine aminonucleoside nephrosis (PAN) rodent model (Grygielko et al, 2005). Briefly, Sprague-Dawley rats were pretreated by oral gavage with 1,3, or 10 mg/kg/day GW788388 once daily.…”

“…The following day, purine aminonucleoside was injected at 15 mg/ 100 g. Treatment groups continued to receive GW788388 for 10 days and then were euthanized. Blood, urine, and kidneys were collected and and kidney mRNA and collagen quantitated exactly as previously described (Grygielko et al, 2005). An additional experiment was conducted in which 300 mg/kg of GW788388 or vehicle control were administered once daily for 4 days to 12 10-week-old Sprague-Dawley rats.…”

Pentoxifylline Attenuates Proteinuria in Anti-Thy1 Glomerulonephritis via Downregulation of Nuclear Factor-κB and Smad2/3 Signaling

“…By using pharmacological inhibitors, Grygielko et al (38) showed that SB525334, a selective ALK5 inhibitor, reduced proteinuria in adriamycin-induced nephrosis in rats. ALK inhibitors act by suppression of TGF-β/Smad signals and inhibition of autoinduction of TGF-β and possibly activin βA (39).…”

Topical application of ALK5 inhibitor A-83-01 reduces burn wound contraction in rats by suppressing myofibroblast population

“…Attempts to inhibit the catalytic activity of ALK5 by using ATP-competitive small molecule inhibitors have been one of the most successful strategies to interfere with the in vivo signaling of TGF-β. These small molecule inhibitors have been proven to prevent kidney and lung fibrosis in animal models [25][26][27] and have shown to possess potent anti-tumor and antimetastatic activity in animal experiments [28][29][30][31][32][33] A-83-01 is also one of the potent small molecule ATP-competitive inhibitors against ALK5 and has been shown to inhibit TGF-β1-dependent cancer metastasis by inhibiting EMT in animals. 19,34) In this report, we attempted to inhibit the TGF-β signaling during burn wound healing in rat skin by using topical application of A-83-01 and evaluated its effect on wound contraction and closing.…”